Growth/differentiation factor of the TGF-β family

ABSTRACT

The invention provides DNA sequences encoding novel members of the TGF-β family of proteins. The TGF-β family comprises proteins which function as growth and/or differentiation factors and which are useful in medical applications. Accordingly, the invention also describes the isolation of the above-mentioned DNA sequences, the expression of the encoded proteins, the production of said proteins and pharmaceutical compositions containing said proteins.

CROSS REFERENCE TO RELATED APPLICATION

This application is a Divisional of Ser. No. 09/901,556 filed Sep. 24,1999 now U.S. Pat. No. 7,067,637; which is a Divisional of Ser. No.08/289,222 filed Aug. 12, 1994, now U.S. Pat. No. 6,120,760; which is aCIP of PCT/EP93/00350 filed Feb. 12, 1993.

The present invention relates to DNA sequences encoding novelgrowth/differentiation factors of the TGF-β family. In particular, itrelates to novel DNA sequences encoding TGF-β-like proteins, to theisolation of said DNA sequences, to expression plasmids containing saidDNA, to microorganisms transformed by said expression plasmid, to theproduction of said protein by culturing said transformant, and topharmaceutical compositions containing said protein. The TGF-β family ofgrowth factors comprising BMP, TGF, and Inhibin related proteins(Roberts and Sporn, Handbook of Experimental Pharmacology 95 (1990),419–472) is of particular relevance in a wide range of medicaltreatments and applications. These factors are useful in processesrelating to wound healing and tissue repair. Furthermore, severalmembers of the TGF-β family are tissue inductive, especiallyosteo-inductive, and consequently play a crucial role in inducingcartilage and bone development.

Wozney, Progress in Growth Factor Research 1 (1989), 267–280 and Vale etal., Handbook of Experimental Pharmacology 95 (1990), 211–248 describedifferent growth factors such as those relating to the BMP (bonemorphogenetic proteins) and the Inhibin group. The members of thesegroups share significant structural similarity. The precursor of theprotein is composed of an aminoterminal signal sequence, a propeptideand a carboxyterminal sequence of about 110 amino acids, which issubsequently cleaved from the precursor and represents the matureprotein. Furthermore, their members are defined by virtue of amino acidsequence homology. The mature protein contains the most conservedsequences, especially seven cysteine residues which are conserved amongthe family members. The TGF-β-like proteins are multifunctional,hormonally active growth factors. They also share related biologicalactivities such as chemotactic attraction of cells, promoting celldifferentiation and their tissue-inducing capacity, such as cartilage-and bone-inducing capacity. U.S. Pat. No. 5,013,649 discloses DNAsequences encoding osteo-inductive proteins termed BMP-2 proteins (bonemorphogenetic protein), and U.S. patent applications Ser. Nos. 179,101and 179,197 disclose the BMP proteins BMP-1 and BMP-3. Furthermore, manycell types are able to synthesize TGF-β-like proteins and virtually allcells possess TGF-β receptors.

Taken together, these proteins show differences in their structure,leading to considerable variation in their detailed biological function.Furthermore, they are found in a wide variety of different tissues anddevelopmental stages. Consequently, they might possess differencesconcerning their function in detail, for instance the required cellularphysiological environment, their lifespan, their targets, theirrequirement for accessory factors, and their resistance to degradation.Thus, although numerous proteins exhibiting tissue-inductive, especiallyosteo-inductive potential are described, their natural role in theorganism and, more importantly, their medical relevance must still beelucidated in detail. The occurrence of still-unknown members of theTGF-β family relevant for osteogenesis or differentiation/induction ofother tissues is strongly suspected. However, a major problem in theisolation of these new TGF-β-like proteins is that their functionscannot yet be described precisely enough for the design of adiscriminative bioassay. On the other hand, the expected nucleotidesequence homology to known members of the family would be too low toallow for screening by classical nucleic acid hybridization techniques.Nevertheless, the further isolation and characterization of newTGF-β-like proteins is urgently needed in order to get hold of the wholeset of induction and differentiation proteins meeting all desiredmedical requirements. These factors might find useful medicalapplications in defect healing and treatments of degenerative disordersof bone and/or other tissues like, for example, kidney and liver.

Thus, the technical problem underlying the present invention essentiallyis to provide DNA sequences coding for new members of the TGF-β proteinfamily having mitogenic and/or differentiation-inductive, e.g.osteo-inductive potential.

The solution to the above technical problem is achieved by providing theembodiments characterized in claims 1 to 17. Other features andadvantages of the invention will be apparent from the description of thepreferred embodiments and the drawings. The sequence listings anddrawings will now briefly be described.

SEQ ID NO. 1 shows the nucleotide sequence of MP-52, i.e. the embryoderived sequence corresponding to the mature peptide and most of thesequence coding for the propeptide of MP-52.

Some of the propeptide sequence at the 5′-end of MP-52 has not beencharacterized so far.

SEQ ID NO. 2 shows the nucleotide sequence of MP-121, i.e. the liverderived sequence corresponding to the mature peptide, the sequencecoding for the propeptide of MP-121, and sequences 5′ and 3′ to thecoding region.

The start codon begins with nucleotide 128 of SEQ ID NO.2. The sequencecoding for the mature MP121 polypeptide begins with nucleotide 836 ofSEQ ID NO. 2. The stop codon begins with nucleotide 1184 of SEQ ID NO.2. The sequence coding for the precursor protein has a length of 1056bp. The sequence coding for the propeptide has a length of 708 bp andthe sequence coding for the mature peptide has a length of 348 bp.

SEQ ID NO. 3 shows the amino acid sequence of MP-52 as deduced from SEQID NO. 1.

SEQ ID NO. 4 shows the amino acid sequence of MP-121 as deduced fromsequence SEQ ID NO.2. The sequence of the mature polypeptide begins withamino acid 237 of SEQ ID NO. 4. The precursor protein has a length of352 amino acids. The propeptide and the mature peptide have a length of236 and 116 amino acids, respectively.

SEQ ID NO. 5 shows a part of the nucleotide sequence of the liverderived sequence of MP-121.

SEQ ID NO. 6 shows a part of the nucleotide sequence of the embryoderived sequence of MP-52.

The shorter DNA-sequences SEQ ID NO. 5 and 6 can be useful for examplefor isolation of further members of the TGF-β-protein family.

FIG. 1 shows an alignment of the amino acid sequences of MP-52 andMP-121 starting from the first of the seven conserved cysteines withsome related proteins. Ia shows the alignment of MP-52 with some membersof the BMP protein family (SEQ ID NOS: 22–27); Ib shows the alignment ofMP-121 with some members of the Inhibin protein family SEQ ID NOS:28–31). * indicates that the amino acid is the same in all proteinscompared; +indicates that the amino acid is the same in at least one ofthe proteins compared with MP-52 (FIG. Ia) or MP-121 (FIG. Ib).

FIG. 2 shows the nucleotide sequences of the oligonucleotide primer asused in the present invention and an alignment of these sequences withknown members of the TGF-β family (SEQ ID NOS: 32–53). M means A or C; Smeans C or G; R means A or G; and K means G or T. 2a depicts thesequence of the primer OD (SEQ ID NOS: 32); 2b shows the sequence of theprimer OID (SEQ ID NOS: 42).

The present invention relates to novel TGF-β-like proteins and providesDNA sequences contained in the corresponding genes. Such sequencesinclude nucleotide sequences comprising the sequence

(SEQ ID NO:7 ATGAACTCCATGGACCCCGAGTCCACA and (SEQ ID NO:8)CTTCTCAAGGCCAACACAGCTGCAGGCACCand in particular sequences as illustrated in SEQ ID Nos. 1 and 2,allelic derivatives of said sequences and DNA sequences degenerated as aresult of the genetic code for said sequences. They also include DNAsequences hybridizing under stringent conditions with the DNA sequencesmentioned above and containing the following amino acid sequences:

(SEQ ID NO:9) Met-Asn-Ser-Met-Asp-Pro-Glu-Ser-Thr or (SEQ ID NO:10)Leu-Leu-Lys-Ala-Asn-Thr-Ala-Ala-Gly-Thr.

Although said allelic, degenerate and hybridizing sequences may havestructural divergencies due to naturally occurring mutations, such assmall deletions or substitutions, they will usually still exhibitessentially the same useful properties, allowing their use in basicallythe same medical applications.

According to the present invention, the term “hybridization” meansconventional hybridization conditions, preferably conditions with a saltconcentration of 6×SSC at 62° to 66° C. followed by a one-hour wash with0.6×SSC, 0.1% SDS at 62° to 66° C. The term “hybridization” preferablyrefers to stringent hybridization conditions with a salt concentrationof 4×SSC at 62°–66° C. followed by a one-hour wash with 0.1×SSC, 0.1%SDS at 62°–66° C.

Important biological activities of the encoded proteins, preferablyMP-52, comprise a mitogenic and osteo-inductive potential and can bedetermined in assays according to Seyedin et al., PNAS 82 (1985),2267–2271 or Sampath and Reddi, PNAS 78 (1981), 7599–7603.

The biological properties of the proteins according to the invention,preferably MP-121, may be determined, e.g., by means of the assaysaccording to Wrana et al. (Cell 71, 1003–1014 (1992)), Ling et al.(Proc. Natl. Acad. of Science, 82, 7217–7221 (1985)), Takuwa et al. (Am.J. Physiol., 257, E797–E803 (1989)), Fann and Patterson (Proc. Natl.Acad. of Science, 91, 43–47 (1994)), Broxmeyer et al. (Proc. Natl. Acad.of Science, 85, 9052–9056 (1988)), Green et al. (Cell, 71, 731–739(1992)), Partridge et al. (Endocrinology, 108, 213–219 (1981)) orRoberts et al. (PNAS 78, 5339–5343 (1981)).

Preferred embodiments of the present invention are DNA sequences asdefined above and obtainable from vertebrates, preferably mammals suchas pig or cow and from rodents such as rat or mouse, and in particularfrom primates such as humans.

Particularly preferred embodiments of the present invention are the DNAsequences termed MP-52 and MP-121 which are shown in SEQ ID Nos. 1 and2. The corresponding transcripts of MP-52 were obtained from embryogenictissue and code for a protein showing considerable amino acid homologyto the mature part of the BMP-like proteins (see FIG. 1 a). The proteinsequences of BMP2 (=BMP2A) and BMP4 (=BMP2B) are described in Wozney etal., Science Vol 242, 1528–1534 (1988). The respective sequences ofBMP5, BMP6 and BMP7 are described in Celeste et al., Proc. Natl. Acad.Sci. USA Vol 87, 9843–9847 (1990). Some typical sequence homologies,which are specific to known BMP-sequences only, were also found in thepropeptide part of MP-52, whereas other parts of the precursor part ofMP-52 show marked differences to BMP-precursors. The mRNA of MP-121 wasdetected in liver tissue, and its corresponding amino acid sequenceshows homology to the amino acid sequences of the Inhibin protein chains(see FIG. 1 b). cDNA sequences encoding TGF-β-like proteins have not yetbeen isolated from liver tissue, probably due to a low abundance ofTGF-β specific transcripts in this tissue. In embryogenic tissue,however, sequences encoding known TGF-β-like proteins can be found inrelative abundance. The inventors have recently detected the presence ofa collection of TGF-β-like proteins in liver as well. The highbackground level of Clones related to known factors of this grouppresents the main difficulty in establishing novel TGF-β-relatedsequences from these and probably other tissues. In the presentinvention, the cloning was carried out according to the method describedbelow. Once the DNA sequence has been cloned, the preparation of hostcells capable of producing the TGF-β-like proteins and the production ofsaid proteins can be easily accomplished using known recombinant DNAtechniques comprising constructing the expression plasmids encoding saidprotein and transforming a host cell with said expression plasmid,cultivating the transformant in a suitable culture medium, andrecovering the product having TGF-β-like activity.

Thus, the invention also relates to recombinant molecules comprising DNAsequences as described above, optionally linked to an expression controlsequence. Such vectors may be useful in the production of TGF-β-likeproteins in stably or transiently transformed cells. Several animal,plant, fungal and bacterial systems may be employed for thetransformation and subsequent cultivation process. Preferably,expression vectors which can be used in the invention contain sequencesnecessary for the replication in the host cell and are autonomouslyreplicable. It is also preferable to use vectors containing selectablemarker genes which can be easily selected for transformed cells. Thenecessary operation is well-known to those skilled in the art.

It is another object of the invention to provide a host cell transformedby an expression plasmid of the invention and capable of producing aprotein of the TGF-β family. Examples of suitable host cells includevarious eukaryotic and prokaryotic cells, such as E. coli, insect cells,plant cells, mammalian cells, and fungi such as yeast.

Another object of the present invention is to provide a protein of theTGF-β family encoded by the DNA sequences described above and displayingbiological features such as tissue-inductive, in particularosteo-inductive and/or mitogenic capacities possibly relevant totherapeutical treatments. The above-mentioned features of the proteinmight vary depending upon the formation of homodimers or heterodimers.Such structures may prove useful in clinical applications as well. Theamino acid sequence of the especially preferred proteins of theTGF-β-family (MP-52 and MP-121) are shown in SEQ ID NO. 3 and SEQ ID NO.4.

It is a further aspect of the invention to provide a process for theproduction of TGF-β-like proteins. Such a process comprises cultivatinga host cell being transformed with a DNA sequence of the presentinvention in a suitable culture medium and purifying the TGF-β-likeprotein produced. Thus, this process will allow the production of asufficient amount of the desired protein for use in medical treatmentsor in applications using cell culture techniques requiring growthfactors for their performance. The host cell is obtainable from bacteriasuch as Bacillus or Escherichia coli, from fungi such as yeast, fromplants such as tobacco, potato, or Arabidopsis, and from animals, inparticular vertebrate cell lines such as the Mo-, COS- or CHO cell line.

Yet another aspect of the present invention is to provide a particularlysensitive process for the isolation of DNA sequences corresponding tolow abundance mRNAs in the tissues of interest. The process of theinvention comprises the combination of four different steps. First, themRNA has to be isolated and used in an amplification reaction usingoligonucleotide primers. The sequence of the oligonucleotide primerscontains degenerated DNA sequences derived from the amino acid sequenceof proteins related to the gene of interest. This step may lead to theamplification of already known members of the gene family of interest,and these undesired sequences would therefore have to be eliminated.This object is achieved by using restriction endonucleases which areknown to digest the already-analyzed members of the gene family. Aftertreatment of the amplified DNA population with said restrictionendonucleases, the remaining desired DNA sequences are isolated by gelelectrophoresis and reamplified in a third step by an amplificationreaction, and in a fourth step they are cloned into suitable vectors forsequencing. To increase the sensitivity and efficiency, steps two andthree are repeatedly performed, at least two times in one embodiment ofthis process.

In a preferred embodiment, the isolation process described above is usedfor the isolation of DNA sequences from liver tissue. In a particularlypreferred embodiment of the above-described process, one primer used forthe PCR experiment is homologous to the polyA tail of the mRNA, whereasthe second primer contains a gene-specific sequence. The techniquesemployed in carrying out the different steps of this process (such asamplification reactions or sequencing techniques) are known to theperson skilled in the art and described, for instance, in Sambrook etal., 1989, “Molecular Cloning: A laboratory manual”, Cold Spring HarborLaboratory Press.

It is another object of the present invention to provide pharmaceuticalcompositions containing a therapeutically-effective amount of a proteinof the TGF-β family of the present invention. Optionally, such acomposition comprises a pharmaceutically acceptable carrier. Such atherapeutic composition can be used in wound healing and tissue repairas well as in the healing of bone, cartilage, or tooth defects, eitherindividually or in conjunction with suitable carriers, and possibly withother related proteins or growth factors. Thus, a therapeuticcomposition of the invention may include, but is not limited to, theMP-52 encoded protein in conjunction with the MP-121 encoded protein,and optionally with other known biologically-active substances such asEGF (epidermal growth factor) or PDGF (platelet derived growth factor).Another possible clinical application of a TGF-β-like protein is the useas a suppressor of the immuno response, which would prevent rejection oforgan transplants. The pharmaceutical composition comprising theproteins of the invention can also be used prophylactically, or can beemployed in cosmetic plastic surgery. Furthermore, the application ofthe composition is not limited to humans but can include animals, inparticular domestic animals, as well. Possible applications of thepharmaceutical composition according to the invention includefurthermore treatment or prevention of connective tissue, skin, mucousmembrane, endothelial, epithelial, neuronal or renal defects, use in thecase of dental implants, use as a morphogenic factor used for inducingliver tissue growth, induction of the proliferation of precursor cellsor bone marrow cells, for maintaining a differentiated state and thetreatment of impaired fertility or for contraception.

Finally, another object of the present invention is an antibody orantibody fragment, which is capable of specifically binding to theproteins of the present invention. Methods to raise such specificantibody are general knowledge. Preferably such an antibody is amonoclonal antibody. Such antibodies or antibody fragments might beuseful for diagnostic methods.

The following examples illustrate in detail the invention disclosed, butshould not be construed as limiting the invention.

EXAMPLE 1 Isolation of MP-121

-   1.1 Total RNA was isolated from human liver tissue    (40-year-old-male) by the method of Chirgwin et al., Biochemistry 18    (1979), 5294–5299. Poly A⁺ RNA was separated from total RNA by oligo    (dT) chromatography according to the instructions of the    manufacturer (Stratagene Poly (A) Quick columns).-   1.2 For the reverse transcription reaction, poly A⁺ RNA (1–2.5 μg)    derived from liver tissue was heated for 5 minutes to 65° C. and    cooled rapidly on ice. The reverse transcription reagents containing    27 U RNA guard (Pharmacia), 2.5 μg oligo d(T)₁₂₋₁₈ (Pharmacia)    5×buffer (250 mM Tris/HCl pH 8.5; 50 mM MgCl₂; 50 mM DTT; 5 mM each    dNTP; 600 mM KCl) and 20 units avian myeloblastosis virus reverse    transcriptase (AMV, Boehringer Mannheim) per μg poly (A⁺) RNA were    added. The reaction mixture (25 μl) was incubated for 2 hours at    42° C. The liver cDNA pool was stored at −20° C.-   1.3 The deoxynucleotide primers OD and OID (FIG. 2) designed to    prime the amplification reaction were generated on an automated    DNA-synthesizer (Biosearch). Purification was done by denaturating    polyacrylamide gel electrophoresis and isolation of the main band    from the gel by isotachophoresis. The oligonucleotides were designed    by aligning the nucleic acid sequences of some known members of the    TGF-β family and selecting regions of the highest conservation. An    alignment of this region is shown in FIG. 2. In order to facilitate    cloning, both oligonucleotides contained EcoR I restriction sites    and OD additionally contained an Nco I restriction site at its 5′    terminus.-   1.4 In the polymerase chain reaction, a liver-derived cDNA pool was    used as a template in a 50 μl reaction mixture. The amplification    was performed in 1×PCR-buffer (16.6 mM (NH₄)₂SO₄; 67 mM Tris/HCl pH    8.8; 2 mM MgCl₂; 6.7 μM EDTA; 10 mM β-mercaptoethanol; 170 μg/ml BSA    (Gibco)), 200 μM each dNTP (Pharmacia), 30 pmol each oligonucleotide    (OD and OID) and 1.5 units Taq polymerase (AmpliTaq, Perkin Elmer    Cetus). The PCR reaction contained cDNA corresponding to 30 ng of    poly (A⁺) RNA as staring material. The reaction mixture was    overlayed by paraffine and 40 cycles (cycle 1: 80 s 93° C./40s 52°    C./40s 72° C.; cycles 2–9: 60s 93° C./40s 52° C./40s 72° C.; cycles    10–29: 60s 93° C./40s 52° C./60s 72° C.; cycles 30–31: 60s 93°    C./40s 52° C./90s 72° C.; cycle 40: 60s 93° C./40s 52° C./420s 72°    C.) of the PCR were performed. Six PCR-reaction mixtures were    pooled, purified by subsequent extractions with equal volumes of    phenol, phenol/chloroform (1:1 (v/v)) and chloroform/isoamylalcohol    (24:1 (v/v)) and concentrated by ethanol precipitation.-   1.5 One half of the obtained PCR pool was sufficient for digestion    with the restriction enzymes Sph I (Pharmacia) and AlwN I (Biolabs).    The second half was digested in a series of reactions by the    restriction enzymes Ava I (BRL), AlwN I (Biolabs) and Tfi I    (Biolabs). The restriction endonuclease digestions were performed in    100 μl at 37° C. (except Tfi I at 65° C.) using 8 units of each    enzyme in a 2- to 12-hour reaction in a buffer recommended by the    manufacturer.-   1.6 Each DNA sample was fractioned by electrophoresis using a 4%    agarose gel (3% FMC Nusieve agarose, Biozym and 1% agarose, BRL) in    Tris borate buffer (89 mM Trisbase, 89 mM boric acid, 2 mM EDTA, pH    8). After ethidiumbromide staining uncleaved amplification products    (about 200 bp; size marker was run in parallel) were excised from    the gel and isolated by phenol extraction: an equal volume of    phenols was added to the excised agarose, which was minced to small    pieces, frozen for 10 minutes, vortexed and centrifuged. The aqueous    phase was collected, the interphase reextracted by the same volume    TE-buffer, centrifuged and both aqueous phases were combined. DNA    was further purified twice by phenol/chloroform and once by    chloroform/isoamylalcohol extraction.-   1.7 After ethanol precipitation, one fourth or one fifth of the    isolated DNA was reamplified using the same 52° C./60s 72° C.; cycle    13: 60s 93° C./40s 52° C./420s 72° C.). The reamplification products    were purified, restricted with the same enzymes as above and the    uncleaved products were isolated from agarose gels as mentioned    above for the amplification products. The reamplification followed    by restriction and gel isolation was repeated once.-   1.8 After the last isolation from the gel, the amplification    products were digested by 4 units EcoR I (Pharmacia) for 0.2 hours    at 37° C. using the buffer recommended by the manufacturer. One    fourth of the restriction mixture was ligated to the vector    pBluescriptII SK+ (Stratagene) which was digested likewise by    EcoR I. After ligation, 24 clones from each enzyme combination were    further analyzed by sequence analysis. The sample restricted by AlwN    I and Sph I contained no new sequences, only BMP6 and Inhibin βA    sequences. 19 identical new sequences, which were named MP-121, were    found by the Ava I, AlwN I and Tfi I restricted samples. The MP-121    containing plasmids were called pSK MP-121 (OD/OID). One sequence    differed from this mainly-found sequence by two nucleotide    exchanges. Ligation reaction and transformation in E. coli HB101    were performed as described in Sambrook et al., Molecular cloning: A    laboratory manual (1989). Transformants were selected by Ampicillin    resistance and the plasmid DNAs were isolated according to standard    protocols (Sambrook et al. (1989)). Analysis was done by sequencing    the double-stranded plasmids by “dideoxyribonucleotide chain    termination sequencing” with the sequencing kit “Sequenase Version    2.0” (United States Biochemical Corporation).

The clone was completed to the 3′ end of the c-DNA by a method describedin detail by Frohman (Amplifications, published by Perkin-ElmerCorporation, issue 5 (1990), pp 11–15). The same liver mRNA which wasused for the isolation of the first fragment of MP-121 was reversetranscribed using a primer consisting of oligo dT (16 residues) linkedto an adaptor primer (AGAATTCGCATGCCATGGTCGACGAAGC(T)₁₆; (SEQ IDNO:11)). Amplification was performed using the adaptor primer(AGAATTCGCATGCCATGGTCGACG (SEQ ID NO:12)) and an internal primer(GGCTACGCCATGAACTTCTGCATA (SEQ ID NO:13) of the MP-121 sequence. Theamplification products were reamplified using a nested internal primer(ACATAGCAGGCATGCCTGGTATTG (SEQ ID NO:14)) of the MP-121 sequence and theadaptor primer. The reamplification products were cloned afterrestriction with Sph I in the likewise restricted vector pT7/T3 U19(Pharmacia) and sequenced with the sequencing kit “Sequenase Version2.0” (United States Biochemical Corporation). Clones were characterizedby their sequence overlap to the 3′ end of the known MP-121 sequence.

One clone, called p121Lt 3′ MP13, was used to isolate a NcoI (bluntended with T₄ polymerase)/SphI fragment. This fragment was ligated intoa pSK MP-121 (OD/OID) vector, where the OD primer sequence was locatedclose to the T7 primer sequence of the pSK+ multiple cloning site,opened with SphI/SmaI. The resulting plasmid was called pMP121DFus6. Itcontains MP-121 specific sequence information starting from position 922and ending with position 1360 of SEQ ID NO. 2.

-   1.9 Using a DdeI fragment of pMP-121DFus6 as a probe, ranging from    nucleotide 931 to nucleotide 1304 of SEQ ID NO. 2, a human liver    cDNA library (Clontech, # HL3006b, Lot 36223) was screened by a    common method described in detail by Ausubel et al. (Current    Protocols in Molecular Biology, published by Greene Publishing    Associates and Wiley-Interscience (1989)). From 8.1×10⁵ phages, 24    mixed clones were isolated and re-screened using the DdeI fragment.    10 clones were confirmed and the EcoRI fragments subcloned into    Bluescript SK (Stratagene, #212206). EcoRI restriction analysis    showed that one clone (SK121 L9.1, deposited by the DSM (#9177) has    an insert of about 2.3 kb. This clone contains the complete reading    frame of the MP121 gene and further information to the 5′ and 3′ end    in addition to the sequence isolated from mRNA by the described    amplification methods. The complete sequence of the EcoRI insert of    SK121 L9.1 is shown in SEQ ID NO.2. The reading frame of the MP-121    gene could be confirmed by sequencing of another clone (SK121    L11.1), having the identical reading frame sequence as SK121 L9.1.    The beginning of the start codon of the MP-121 sequence of SK121    L9.1 could be determined at position 128 of SEQ ID NO.2, since there    are three stop codons in-frame in front of the start codon at    positions 62, 77 and 92. The start site of the mature MP-121 is at    position 836 of SEQ ID NO.2 in sequence analogy to other members of    the TGF-β-family, corresponding to amino acid 237 in SEQ ID NO.4.    The stop codon is at position 1184 of SEQ ID NO.2.

Plasmid SK121 L9.1 was deposited under number 9177 at DSM (DeutscheSammlung von Mikroorganismen und Zellkulturen), Mascheroder Weg 1b,Braunschweig, on 26.04.94).

EXAMPLE 2 Isolation of MP-52

A further cDNA sequence, MP-52, was isolated according to the abovedescribed method (Example 1) by using RNA from human embryo (8–9 weeksold) tissue. The PCR reaction contained cDNA corresponding to 20 ng ofpoly (A⁺)RNA as starting material. The reamplification step was repeatedtwice for both enzyme combinations. After ligation, 24 clones from eachenzyme combination were further analyzed by sequence analysis. Thesample restricted by AlwN I and Sph I yielded a new sequence which wasnamed MP-52. The other clones comprised mainly BMP6 and one BMP7sequence. The sample restricted by Ava I, AlwN I and Tfi I contained nonew sequences, but consisted mainly of BMP7 and a few Inhibin βAsequences.

The clone was completed to the 3′ end according to the above describedmethod (Example 1). The same embryo mRNA, which was used for theisolation of the first fragment of MP-52, was reverse transcribed as inExample 1. Amplification was performed using the adaptor primer(AGAATTCGCATGCCATGGTCGACG (SEQ ID NO:12)) and an internal primer(CTTGAGTACGAGGCTTTCCACTG (SEQ ID NO: 15)) of the MP-52 sequence. Theamplification products were reamplified using a nested adaptor primer(ATTCGCATGCCATGGTCGACGAAG (SEQ ID NO: 16)) and a nested internal primer(GGAGCCCACGAATCATGCAGTCA (SEQ ID NO:17)) of the MP-52 sequence. Thereamplification products were cloned after restriction with Nco I in alikewise restricted vector (pUC 19 (Pharmacia #27-4951 -01) with analtered multiple cloning site containing a unique Nco I restrictionsite) and sequenced. Clones were characterized by their sequence overlapto the 3′ end of the known MP-52 sequence. Some of these clones containthe last 143 basepairs of the 3′ end of the sequence shown in SEQ ID NO:1 and the 0.56 kb 3′ non translated region (sequence not shown). One ofthese was used as a probe to screen a human genomic library (Stratagene#946203) by a common method described in detail by Ausubel et at.(Current Protocols in Molecular Biology, published by Greene publishingAssociates and Wiley-Interscience (1989)). From 8×10⁵ λ phages one phage(A 2.7.4) which was proved to contain an insert of about 20 kb, wasisolated and deposited by the DSM (#7387). This clone contains inaddition to the sequence isolated from mRNA by the describedamplification methods sequence information further to the 5′ end. Forsequence analysis a Hind III fragment of about 7,5 kb was subcloned in alikewise restricted vector (Bluescript SK, Stratagene #212206). Thisplasmid, called SKL 52 (H3) MP12, was also deposited by the DSM (#7353).Sequence information derived from this clone is shown in SEQ ID NO: 1.At nucleotide No. 1050, the determined cDNA and the respective genomicsequence differ by one basepair (cDNA: G; genomic DNA: A). We assume thegenomic sequence to be correct, as it was confirmed also by sequencingof the amplified genomic DNA from embryonic tissue which had been usedfor the mRNA preparation. The genomic DNA contains an intron of about 2kb between basepairs 332 and 333 of SEQ ID NO: 1. The sequence of theintron is not shown. The correct exon/exon junction was confirmed bysequencing an amplification product derived from cDNA which comprisesthis region. This sequencing information was obtained by the help of aslightly modified method described in detail by Frohman (Amplifications,published by Perkin-Elmer Corporation, issue 5 (1990), pp 11–15). Thesame embryo RNA which was used for the isolation of the 3′ end of MP-52was reverse transcribed using an internal primer of the MP-52 sequenceoriented in the 5′ direction (ACAGCAGGTGGGTGGTGTGGACT (SEQ ID NO: 18)).A polyA tail was appended to the 5′ end of the first strand cDNA byusing terminal transferase. A two step amplification was performed firstby application of a primer consisting of oligo dT and an adaptor primer(AGAATTCGCATGCCATGGTCGACGAAGC(T₁₆)(SEQ ID NO:11)) and secondly anadaptor primer (AGAATTCGCATGCCATGGTCGACG (SEQ ID NO: 12)) and aninternal primer (CCAGCAGCCCATCCTTCTCC (SEQ ID NO: 19)) of the MP-52sequence. The amplification products were reamplified using the sameadaptor primer and a nested internal primer (TCCAGGGCACTAATGTCAAACACG(SEQ ID NO: 20)) of the MP-52 sequence. Consecutively thereamplification products were again reamplified using a nested adaptorprimer (ATTCGCATGCCATGGTCGACGAAG (SEQ ID NO: 16)) and a nested internalprimer (ACTAATGTCAAACACGTACCTCTG (SEQ ID NO: 21)) of the MP-52 sequence.The final reamplification products were blunt end cloned in a vector(Bluescript SK, Stratagene #212206) restricted with EcoRV. Clones werecharacterized by their sequence overlap to the DNA of λ2.7.4.

Plasmid SKL 52 (H3) MP12 was deposited under number 7353 at DSM(Deutsche Sammlung von Mikroorganismen und Zellkulturen), MascheroderWeg 1b, 3300 Braunschweig, on 10.12.1992.

Phage λ 2.7.4. was deposited under number 7387 at DSM on 13.1.1993.

1. An isolated protein of the TFG-β-family selected from the groupconsisting of: (a) a protein comprising the amino acid sequence shown inSEQ ID NO:22, wherein said protein has essentially the same cartilage orbone inducing activities as a mature protein encoded by the nucleotidesequence of SEQ ID NO:1, and (b) a protein consisting of a fragment ofthe amino acid sequence of SEQ ID NO:3, wherein said fragment comprisesthe amino acid sequence shown in SEQ ID NO:22.
 2. A pharmaceuticalcomposition comprising a at least one protein according to claim 1 and apharmaceutically acceptable excipient.
 3. The pharmaceutical compositionaccording to claim 2 further comprising a growth/differentiation factorselected from the group consisting of MP-121, epidermal growth factorand platelet derived growth factor.